Overexpression of autocrine TGF-β1 suppresses the growth of spindle epithelial cells in vitro and in vivo in the rat 4NQO model of oral carcinogenesis

Abstract

We examined the effect of the stable transfection of latent TGF-β1 cDNA, under the control of a cytomegalovirus promoter in the expression vector pcDNA3, into a 4NQO-induced clonal rat oral keratinocyte cell line that formed undifferentiated spindle cell tumours following subcutaneous transplantation to athymic mice. Test cells containing latent TGF-β1 cDNA produced a 2.3-fold increase in TGF-β1 protein compared to pcDNA3 controls as demonstrated by ELISA. Neutralisation experiments indicated that the majority of the protein was in the latent form. Untransfected and transfected (containing either TGF-β1 cDNA or pcDNA3) cell lines were keratin negative and vimentin positive. Cells transfected with TGF-β1 were inhibited more than pcDNA3 controls when cultured in an anchorage dependent or independent environment. Subcutaneous transplantation of cells overproducing TGF-β1 resulted in tumours of significantly smaller volume than vector-only controls. Further, orthotopic transplantation of cells containing TGF-β1 cDNA to the floor of the mouth in athymic mice markedly inhibited the development of pulmonary metastases compared to vector only controls. Both test and control cell lines in athymic mice formed undifferentiated tumours with a complete absence of keratin elaboration. Subcutaneous xenografts were recultured and cells containing the TGF-β1 cDNA produced a similar amount of TGF-β1 peptide as the cells containing pcDNA3 only. The production of TGF- β1 by both of the xenograft-derived cell lines was significantly less than the parent, pre-transplanted cell lines and the untransfected cell line. All of the cell lines were inhibited by exogenous TGF-β1. Our results demonstrate that autocrine TGF-β1 functions as a tumour suppressor in vitro and in vivo in 4NQO-induced spindle tumour cells that are growth inhibited by the ligand. Furthermore, tumour formation in athymic mice is associated with selection for a cell phenotype with diminished autocrine TGF-β1 production.